Soybean (genus Glycine) belongs to the Fabaceae family (leguminosae), as well as bean (Phaseolus), lentil and pea (Pissum), and is a protein-rich grain, grown as food, both for human and animals. The word soy comes from Japanese shoyu and originated in China. The Fabaceae family is one of the largest plant families, also known as Leguminosae, having a broad geographic distribution. There are approximately 18,000 species in more than 650 genera. The typical fruit of this type of family is the legume, also known as pod (there are exceptions). It is subdivided into 3 very different subfamilies: Faboideae (or Papilionoideae), Caesalpinioideae (or Caesalpiniaceae) and Mimosoideae (or Mimosaceae). The variation in name is due to the current coexistance of more than one classification system. The literature describes that the roots of almost all species in this family live in symbiosis with bacteria of the Rhizobium genus and the like, responsible for nitrogen fixation in the air, a relevant and significant ecological characteristic. Furthermore, they are of high economic value for food production.
Soybean is considered an important crop and is highly valued by world agriculture. In this sense, one of the major objectives of the soybean breeders is to develop more stable, productive and disease-resistant varieties, such as, rust-resistant varieties, for instance. One of the main reasons for this concern is to maximize grain yield for human and animal consumption. In order to attain said objects, the breeder should select and develop cultivars having superior traits over those available in the market.
Borém (1998) defines improvement as “the art and science that aim at the genetic modification of plants to render them more useful to men”. Breeder Manoel Abílio de Queiroz (2001) (Queiroz, M. A., 2001 “Melhoramento Genético no Brasil—realizações e perspectivas”. In: Recurso Genéticos e Melhoramento de Plantas, cap.1. pg. 1-28.) comments on this respect on a broader perspective, contemplating the entire development of agriculture in the last ten thousand years, and its role in changing the habits of human populations that abandoned nomadism and adopted sedentarism. This happened when people decided to abandon extractivism to start growing crops that were more suitable for their survival. This practice has lasted for a long period and caused changes in the gene frequencies of the chosen species, bringing significant benefits to the world agriculture. It is known that the domestication of new species is relatively recent and very limited, and that most species that feed humanity were domesticated in remote times, particularly grains.
The same author reports that with the advent of genetic improvement after the discovery of Mendel's Laws, science started to play a leading role in the development of significant crops for human consumption. In this respect, texts were published in the most different areas of knowledge and in different branches of genetics, including DNA genetic markers, which are relevant for monitoring genetic plant improvement with a view to making it more efficient in the search for enhancing traits that may meet society's needs.
The enhancement of plants aims at obtaining plant varieties that are superior to those available in the market, either in the production of grains, green mass or fibers, in resistance to pests and diseases, or in higher protein and oil content, fiber quality or other traits of interest (Conagin, A.; Ambrosano, G. M. B.; Nagai, V. Poder discriminativo da posição de classificação dos testes estatísticos na seleção de genótipos. Bragantia, v. 56, no. 2, p. 403-417, 1997).
Among the main contributions of the soybean improvement in Brazil is the development of varieties capable of adapting to lower latitudes and resistant to major diseases (Arias, C. A. A. et al. Melhoramento e Biotecnologia: ferrugem da soja. In: IV Brazilian Soybean Congress held on Jun. 5-8, 2006. Londrina (Proceedings)). The same authors also report that studies involving genetic resistance provided positive results in helping to solve disease problems. Some of the diseases include: frogeye leaf spot (Cercospora sojina), soybean stem canker (Diaporthe phaseolorum f.sp. meidionallis), powdery mildew (Microsphaera diffusa) and soybean cyst nematode (Heterodera glycines). In addition, Brogin (2005), (Brogin, R. L. Mapeamento de genes de resistência à ferrugem e de QTL's envolvidos na resistência à Septoriose. 2005. Dissertation (Doctorate in Genetics and Plant Improvement—Escola Superior de Agricultura “Luiz de Queiroz”, E-SALQ/USP, Piracicaba, S P.) described studies on resistance to bacterial pustule, bacterial blight, brown stem rot, stem necrosis virus and soybean mosaic virus.
In the case of soybean, it is known that the improvement has been made by the introduction of materials, selection and hybridization (artificial crossbreeding), resulting in a new pure line gathering favorable alleles present in two or more genotypes. The material resulting from these processes can be used by rural producers as a new cultivar.
It is known that the success of a new variety depends on the choice of germplasm, of a good crossbreeding block planning to be used by the breeder to attain the desired objectives, in addition to the selection of parents. It is also known that there is a need to know the type of trait inheritance so as to rationalize the cultivar development process (Arias, C. A. A. et al. Melhoramento e Biotecnologia: ferrugem da soja. In: IV Brazilian Soybean Congress held on Jun. 5-8, 2006. Londrina (Proceedings)). The ability to predict the inheritance that certain traits will provide is essential in agriculture. Traits controlled by a single gene show results expected by Mendel's principles. However, traits controlled by more than one locus may differ from the expected results. Statistic methods and experimental projects are created in order to predict the inheritance of several quantitative traits related to phenotypic traits.
In conducting segregating generations, several methods are used, among which are the Bulk Method, SSD (Single Seed Descent) and backcrossing. In the bulk method, segregating generations, generally F2 and F5, are grown with the seeding and harvest of all the plants mixed in a single population. Therefore, in the bulk method, the seeds used for growing each segregating generation are a sample of the seeds harvested in the previous generation. After five generations of self-fertilizing crops, the plants exhibit a high degree of homozygosis and can be selected for individual harvest (Souza, A. P. Biologia Molecular Aplicada ao Melhoramento. In: Recursos Genéticos e Melhoramento—Plantas. Luciano L. Nass; Afonso C. C. Valois; Itamar S. de Melo; Maria Clélia Valadares-Inglis. (Org.) 1a. Ed. Rondonópolis, 2001, v. 1, p 939-966.
The SSD method in soybean was described by Brim (1966) (Brim, C. A.; 1966. A modified pedigree method of selection in soybeans. Crop Science, v. 6, p. 20) and consists of segregating generation advancement (from F2 to F5) harvesting a single pod (2 to 3 seeds) from each plant; however, only one plant from each pod is used to grow the next generation. A sample is harvested and conserved. In his way, at the end of the process, each line corresponds to a different F2 plant and, therefore, there is a reduction in the loss caused by deficient sampling or natural selection.
Backcrossing is not exactly a method for growing segregating populations. It is a strategy used to improve the phenotypic expression of a deficient trait, especially if this trait is of a qualitative inheritance. The use enables the transfer of a gene or of a few genes from a parent called donor parent (DP) to another parent called recurrent parent (RP), and the recurrent parent is usually a cultivar of commercial interest having some kind of deficiency in its cultivation that needs to be improved. This deficiency can be corrected by the process of transferring the gene from the donor parent, which does not have the deficiency, to the recurrent parent. This procedure, that is to say, the cross of individuals from the segregating population with the recurring parent, is called backcrossing and is responsible for recovering almost 100% of the recurring parent genotype (Souza, A. P. Biologia Molecular Aplicada ao Melhoramento. In: Recursos Genéticos e Melhoramento—Plantas. Luciano L. Nass; Afonso C. C. Valois; Itamar S. de Melo; Maria Clélia Valadares-Inglis. (Org.) 1a. Ed. Rondonópolis, 2001, v. 1, p 939-966).
In the end of the selective process, the breeder identifies one or a few pure lines with superior traits that will originate a new cultivar.
It is important to point out that in terms of improvement aiming at disease and pest resistance, gene pyramiding is recommended. Pyramiding relates to the association of several resistance genes present in a same cultivar aiming at obtaining a lasting and broad-spectrum resistance (Kelly, J. D.; Gepts, P.; Miklas, P. N.; Coyne, D. P. Tagging and mapping of genes and QTL and molecular marker-assisted selection for traits of economic importance in bean and cowpea. Field Crops Research, v. 82, p. 135-154, 2003).
However, in practice, the pyramiding of resistance genes or even rust tolerance (ASR) is difficult because the breeder cannot visually distinguish the plants with one or more resistance or tolerance genes, since phenotypically they have the same kind of reaction, that is, RB lesions.
In this context, biotechnology arose as a tool to facilitate and accelerate research in widely different areas, in particular genetics and improvement. The use of molecular markers is an effective and rapid strategy in the identification and transfer of new genes (Tanskley S.D. (1983). Molecular markers in plant breeding. Plant Molecular Biology Rep. 1: 3-8; Tanskley, S. D., McCouch S. R. (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science v. 277: 1063-1066). Molecular markers can be used in improvement programs directed to selecting qualitative and quantitative traits.
It is known that molecular markers appeared as a huge contribution from the development of molecular techniques that enable genome analysis. Molecular markers can have many uses in plants, being especially utilized in gene mapping and QTLs (“Quantitative Trait Loci”) of interest. Song et al. (2004) (Song, Q. J., Marek, L. F., Shoemaker, R. C., Lark, K. G., Concibido, V. C., Delannay, X., Specht, J. E., Cregan, P. B. (2004). A new integrated genetic linkage map of the soybean. Theoretical and Applied Genetics, v. 109, p. 122-128), have developed a dense soybean linkage map, and it is known that the association of genes with molecular markers in soybean is being widely used.
The following examples of molecular markers can be mentioned: isoenzyme electrophoresis, “Restriction Fragment Length Polymorphisms” (RFLPs), “Random Amplified Polymorphic DNAs” (RAPDs), “Arbitrarily Primed Polymerase Chain Reaction” (AP-PCR), “DNA Amplification Fingerprinting” (DAF), “Sequence Characterized Amplified Regions” (SCARs), “Amplified Fragment Length Polymorphisms” (AFLPs), “Simple Sequence Repeats” (SSRs) and “Single Nucleotide Polymorphisms” (SNPs). Among the molecular markers cited above, SSRs are of interest for genetic mapping because each marker corresponds to a single position in the genome, but has several alleles yielding a high degree of polymorphism (Cregan P. B., Jarvik, T., Bush, A. L., Shoemaker, R. C., Lark, K. G., Kahler, A. L., Kaya, N., VanToai, T. T., Lohnes D. G., Chung, J., Specht, J. E. (1999). An integrated genetic linkage map of the soybean genome. Crop Science, v. 39: 1464-1490). Furthermore, they are easy-to-use, yield consistent results and are accessible to almost all biotechnology labs.
The construction of a genetic map requires the definition of the types of markers to be mapped and the type of genetic delineation to be used to detect the existing linkage disequilibrium among them. The several types of genetic delineations that can be used to construct the genetic map of plant species have in common the yield of generations showing linkage disequilibrium for segregating loci, enabling linkage analysis. Several factors are responsible for linkage disequilibrium, among them genetic selection and derivation. However, in segregating generations derived from crossings between lines (for instance, generation F2 and backcrossing), the main cause for this is related to the physical linkage of the loci, having been the genetic basis of the classical linkage analysis constructed. Due to the physical linkage of the loci, linkage disequilibrium is elevated in the populations derived from controlled crosses and, consequently, the ability to detect the linkage between two physically linked loci is also high. Therefore, the suitable choice of parent materials is essential in the first stage of the process (Coelho, A. S. G. e Silva, H. D. “Construção de Mapas Genéticos & Mapeamento de QTL's”. Piracicaba, S P—February, 2002. 66 p).
After these steps, linkage analysis is carried out with a view to generate a genetic map involving an assessment of the segregation pattern of individual markers, the detection of the linkage disequilibrium between pairs of markers, the measuring of the distance between markers and the ordering of markers in linear linkage groups. The phenotypical class for which the gene is responsible is also considered a marker; however, it is a morphological marker, rather than a molecular marker (Coelho, A. S. G e Silva, H. D. 2002—cited above).
According to the same authors, the detection of the linkage between the loci is performed based on the X2 adherence test, which is based on the comparison between the frequencies observed in the different genotypic classes and those expected under the independent segregation condition between loci. This test provides an estimate of the likelihood of observed deviations given the independence condition. The 5% likelihood level is the critical point for rejecting the independence hypothesis. In the conditions wherein the likelihood of deviations found, given the independence condition, was lower than 5%, the hypothesis was rejected and it is considered that the loci are not independently segregating, that is, they are linked.
In the present invention, as there already is a soybean consensus linkage map, gene-linked markers were identified through the BSA (Bulked Segregant Analysis) method. The BSA method, proposed by Michelmore et al. (1991), (Michelmore, R. W., Paran, I., Kessell, R. V. (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of National Academy of Science of USA, v. 88, p. 9828) is a quick and efficient strategy for identifying the link between molecular markers and qualitative traits. By using this technique, individuals from a F2 population segregating for 2 alleles of a single gene are grouped in 2 bulks of contrasting homozygous genotypes. Since the division of bulks is made taking into account only the trait of interest, all the other genes are randomly grouped. Thus, the genetic background becomes equal between the 2 groups and they only differ in the selected region. As a consequence, a polymorphic marker between the contrasting parents having the same polymorphism between the 2 bulks is very likely to be associated with the gene of interest. The authors emphasize that the markers need to be closer than 15 cM from the locus of interest to be effective, because at this distance, even if recombination occurs, the marker and the gene tend to remain together. However, the threshold distance for detecting the linkage seems to be 25 cM.
For the construction of bulks, the minimum number of individuals used is determined by the frequency with which polymorphism of markers unlinked to the gene of interest is expected to be found between the bulks, and this is reflected in the type of marker used (dominant or codominant). For the dominant marker, for instance, the likelihood of a bulk of n individuals having a band and a second bulk with the same number of individuals not having this band is 2(1−[¼n])(¼)n when the marker is unlinked to the target gene. That is, for 2 contrasting bulks of 10 individuals, the probability of a marker unlinked to the target gene being polymorphic between 2 bulks is of 2×10−6. Therefore, with a few individuals in each bulk and even when several markers are used, the chances of detecting unlinked markers are small (Michelmore, R. W., Paran, I., Kessell, R. V. (1991).
Mapmaker (Lander E. S.; Green, P.; Abrahamson, J.; Barlow, A.; Daly, M. J.; Lincoln, S. E.; Newburg, L. (1987). Mapmaker: an interactive computer package for constructing genetic linkage maps of experimental and natural populations. Genomics, v. 1, p. 174-181) is an interactive software for constructing genetic linkage maps. The program uses an efficient algorithm to carry out multipoint linkage analyses (which is a simultaneous estimate of all the recombination fractions in the data) of several loci, working with dominant, recessive or codominant molecular markers. The data used can originate from backcrossing, F2 or F3 populations resulting from interbreeding and recombinant inbred lines. The distances between the loci are calculated using likelihood.
The construction of linkage maps is hampered by genotyping errors. Low error rates cause an expansion of the map and interfere in determining the correct gene order. That is why the current version of MapMaker/EXP 3.0 (Lincoln, S. E., Lander, S. L. (1993). Mapmaker/exp 3.0 and Mapmaker/QTL 1.1 Whitehead Inst. Of Med Res. Tech Report. Cambridge, Mass.) incorporated an algorithm for detecting potential genotyping errors (Lincoln, S. E.; Lander, S. L. (1992) Systematic detection of errors in genetic linkage data. Genomics, v. 14, p. 604-610). The method detects the majority of errors and with this function it is possible to construct accurate maps.
In the screening process for disease resistance, especially when the pathosystems are difficult to work with due to maintenance difficulties, isolation and pathogen inoculation, or in the disease assessment process, the use of molecular markers is highly recommended. This is the case with Asian soybean rust. It is known that molecular markers detect the genetic information without environmental interference. Another aspect to be considered is the detection of variations in the nucleotide sequence and even in the nontranscribed regions. In this way, it is possible to eliminate and relieve the need to use work-intensive phytopathological processes, identifying individuals from a segregating population carrying the marker linked to the favorable allele of interest, resulting in time and resource savings.
Within this context, the present invention is a significant contribution to improvement programs directed to the use of molecular marker assisted screening, using molecular markers linked to disease-resistant genes, particularly resistant to Asian soybean rust. These markers help to position genes related with disease resistance in the soybean linkage map, for instance.
Asian soybean rust (ASR), caused by fungus Phakopsora pachyrhizi, is considered the most destructive soybean leaf disease (Miles, M. R.; Frederick, R. D.; Hartman, G. (2003) Soybean rust: Is the U.S. soybean crop at risk? Online. APSnet Feature, American Phytopathological Society). The disease spreads through uredospores and has the potential of causing severe damages to soybean crops.
Discovered in Japan in 1902, it has spread to Asia and Australia in 1934, (Kochman, J. K. (1977). Soybean rust in Australia. Pp. 44-48 In: Rust of Soybean—The problem and research needs. R. E. Ford and J. B. Sinclair, eds. International Agricultural Publications, Manila, The Philippines), India in 1951 (Sharma, N.D.; Mehta, S. K. (1996). Soybean rust in Madhya Pradesh. Acta Botanica Indica, v. 24: 115-116), Hawaii in 1994 (Killgore, E.; Heu, R. (1994). First report of soybean rust in Hawaii. Plant Disease, v. 78: 1216), and Africa in 1996 (Akinsanmi, O. A.; Ladipo, J. L.; Oyekan, P. O. (2001). First report of soybean rust (Phakopsora pachyrhizi) in Nigeria. Plant Disease, v. 85, p. 97). In South America, the disease was reported for the first time in 2001 in Paraguay (Morel, W.; Yorinori, J. T. (2002). Situacion de la roja de la soja em el Paraguay. Bol de Divulgacion No. 44. Ministério da Agricultura y Granaderia, Centro Regional de Investigacion Agrícola, Capitan Miranda, Paraguay) and in the following years it arrived in Brazil, Argentina, Bolivia and Colombia (Rossi, R. L. (2003). First report of Phakospora pachrhizi, the casual organism of soybean rust in the Provence of Misiones, Argentina. Plant Disease, v. 87: 102; Yorinori, J. T.; Lazzarotto, J. J. (2004). Situação da ferrugem asiatica da soja no Brasil e na América do Sul. In: Documentos/Embrapa Soja, no. 236, Londrina). In these countries, the losses in productivity due to Asian soybean rust were drastic, varying from 10% to 80% in some crops (Yorinori J T (2004) Ferrugem “asiática” da soja no Brasil: evoluçáo, importância econômica e controle. In: Junior J N, Lazzarotto J J (eds) Documentos 247. Embrapa, Londrina, Brazil, 36p. In the United States, the first symptoms of the disease were reported in November, 2004 (Schneider, R. W.; Hollier, C. A.; Hitam, H. K. (2005). First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States. Plant Disease, v. 89: 774), and, in 2005, yield loss was estimated to be up to 50% (Dorrance, A. E.; Draper, M. A.; Hershman, D. E., eds. Using Foliar Fungicides to Manage Soybean Rust. NC-504 Land Grant Universities Cooperating. Bulletin SR-2005). However, due to unfavorable environmental conditions for the pathogen, the disease did not achieve the expected levels (Sconyers, L. E.; Kemerait, R. C.; Brock, J.; Phillips, D. V.; Jost, P. H.; Sikora, E. J.; Gutierrez-Estrada, A.; Muller, J. D.; Marois, J. J.; Wright, D. L.; Harmon, C. L. (2006). Asian soybean rust development in 2005: A perspective from the Southeastern United State Online. APSnet Feature, American Phytopathological Society).
The development of the fungus is favored by temperatures between 15° and 29° C. and high humidity. In favorable conditions, the symptoms can be detected in 5 to 8 days after the plant is infected by the uredospores (Marchett, M. A.; Melching, J. S.; Bromfield, K. R. (1976). The effects of temperature and dew period on germination and infection by uresdospores of Phakopsora pachyrhizi. Phytopathology, v. 66: 461-463; Melching, J. S.; Dowler, W. M.; Koogle, D. L.; Royer, M. H. (1989). Effects of duration, frequency, and temperature of leaf wetness periods on soybean rust. Plant Disease, v. 73: 117-122). In cultivated soybean, the first symptoms are light-brown polygonal lesions of 2 to 5 mm on the adaxial leaf surface. In the 10 to 14 days period, volcano-shaped lesions known as pustules appear on the abaxial surface of the leaf, where uredospores are produced (Marchett, M. A.; Uecker, F. A.; Bromfield, K. R. (1975). Uredial development of Phakopsora pachyrhizi in soybeans. Phytopathology, v. 65: 822-823.; Melching, J. S.; Dowler, W. M.; Koogle, D. L.; Royer, M. H. (1989). Effects of duration, frequency, and temperature of leaf wetness periods on soybean rust. Plant Disease, v. 73: 117-122).The effects of temperature and dew period on germination and infection by uresdospores of Phakopsora pachyrhizi. Phytopathology, v. 66: 461-463; Melching, J. S.; Dowler, W. M.; Koogle, D. L.; Royer, M. H. (1989). Effects of duration, frequency, and temperature of leaf wetness periods on soybean rust. Plant Disease, v. 73: 117-122). As the infection increases, severe lesions and premature defoliation occur in the plants.
Although fungicides minimize losses, the use of resistant or tolerant cultivars is the best alternative for controlling the disease, in order to reduce costs, facilitate management and help in environmental conservation. It is known that fungus resistance naturally occurs in genotypes of the Glycine genus (Burdon, J. J.; Marshall, D. R. (1981). Evaluation of Australian native species of Glycine canescens, a wild relative of soybean. Theoretical Applied Genetics, v. 65: 44-45; Burdon, J. J. (1988). Major gene resistance to Phakopsora pachirhizi in Glycine canescens, a wild relative of soybean. Theoretical Applied Genetis, v. 75: 923-928), and is typically conferred by the hypersensitivity reaction. This is a common type of immune response caused by the presence of resistance genes (R-gene) of the plant when challenged by pathogen avirulence genes (Avr-genes) (McDowell, J. M.; Simon, S. A. (2006). Recent insights into R gene evolution. Molecular Plant Pathology, v. 7: 437-448). In this sense, resistant genotypes show a reddish-brown (RB) lesion with no or little sporulation, while susceptible genotypes show a light-brown (TAN) lesion and profuse sporulation.
During the process for the development of a ASR-resistant or -tolerant soybean variety, the plants have to be assessed at each generation as to their level of resistance or tolerance to the pathogen, thus identifying the resistant or tolerant plants at each cycle, until the superior cultivar is selected. In practice, the infection process occurs naturally when the fungi spores are in the air or artificially by spraying the leaves with a spore solution collected from previously infected plants. Natural occurrence is cyclic and very dependent on climate conditions. Furthermore, the breeder not always has available spores to promote artificial inoculations. The reasons range from technical difficulties to keeping the infected plants and legal prohibitions. This difficulty is more pronounced when trying to develop resistant cultivars in countries where the disease has not occurred yet and where the pest is still quarantinable.
Cultivated soybean (Glycine max) has four qualitative dominant resistance genes. Rpp1 identified in PI 200492 ((McLean, R. J.; Byth, D. E. (1980). Inheritance of resistance to rust (Phakopsora pachyrhizi) in soybean. Australian Journal Agricultural Research, v. 31; 951-956); Rpp2 na PI 230970 (Bromfield, K. R.; Hartwig, E. E. (1980). Resistance to soybean rust and mode of inheritance. Crop Science, v. 20, n. 2, p. 254-255); Rpp3 in PI 462312 (Bromfield, K R.; Melching, J. S. (1982). Sources of specific resistance to soybean rust. (Abstr.) Phytopatology, v. 72, p. 706), and Rpp4 in PI 459025 (Hartwig, E. E. (1986). Identification of a fourth major gene conferring to resistance to soybean rust. Crop Science, v. 26, p. 1135-1136). The resistance presented by each gene is limited to the specific pathogen variety (Bonde, M. R.; Nester, S. E.; Austin, C. N.; Stone, C. L.; Frederick, R. D.; Hartman, G. L.; Miles, M. R. (2006). Evaluation of virulence of Phakopsora pachyrhizi and P. meibomiae isolates. Plant Disease, v. 90, p. 708-716) and this resistance can be overcome in a short period of time due to the coevolution of host resistance and pathogen virulence (McDonald, B. A.; Celeste, L. (2002). Pathogen population genetics, evolutionary potential, and durable resistance. Annual Ver. Phytopathology, v. 40, p. 349-379). Additionally, the fungus has different varieties with variable geographic distribution. Therefore, it is impossible, for instance, to ensure that an ASR-resistant or -tolerant cultivar selected with spores produced under field conditions in the USA will be resistant or tolerant when cultivated under field conditions in Brazil.
In 2002, the resistance provided by the FT-2 cultivar and the four ASR-resistance genes were previously reported in Brazil as being effective (Arias, C. A. A.; Brogin, R. L.; Yorinori, J. T.; Kiihl, R. A. de S.; Toledo, J. F. F. (2003). Um gene dominante determinando a resist{tilde over (e)}ncia da cultivar FT-2 à ferrugem da soja (Phakopsora pachyrhizi Sydow). In: Congresso Brasileiro de Melhoramento de Plantas, 2; Porto Seguro, 2003. Proceedings. Porto Seguro: Sociedade Brasileira de Melhoramento de Plantas—SBMP—. (Compact disc)). However, in the next harvest, the Rpp1, Rpp3 and FT-2 resistance was simultaneously broken ((Yorinori, J. T. (2004). Ferrugem “asiática” da soja no Brasil: evolução, importância economica e controle. Yorinori, J. T. et al. (eds)—Londrina: Embrapa Soja, 2004, 36 p. (Documentos, 27)). Thus, the efforts to control ASR with the use of resistant cultivars carrying single genes have not yet proved to be successful. Therefore, the identification and use of new disease resistance sources are pursued by the genetic improvement programs carried out by geneticists and soybean breeders, and are essential for those who are involved with soybean.
In view of the foregoing, the importance of the present invention is clear as a highly applicable methodology to help in the control of ASR, facilitating and accelerating the development of disease-resistant or -tolerant cultivars. The present invention solves the problem of the diversity in varieties and the geographic distribution of the pathogen because it enables the selection to be made in the absence of the pathogen by analyzing DNA polymorphic markers linked to rust resistance or tolerance alleles.
The present invention discloses the identification of five new sources of ASR resistance by means of genetic analyses. There are two cases with dominant resistance (PI 200487 and PI 200526), in two of the sources, resistance is recessive (PI 200456 e PI 224270) and, in the other, resistance has incomplete dominance (PI 471904). It is the first case of ASR resistance conditioned by a recessive gene/allele.
In addition, the present invention also uses SSR molecular markers from the soybean linkage map to construct the genetic map of the genes of these new sources, as well as the map of the genes present in the original sources of Rpp2 (PI 230970) and Rpp4 (PI 459025).
The present invention enables the association of resistance or tolerance genes, because it identifies not only new loci and alleles associated with these loci, but also DNA segments linked to said loci that can be easily tracked during the genetic improvement process, regardless of the improvement method used by the breeder (SSD, Bulk, Backcrossing, etc.), using routine DNA analysis techniques.
In the context of the present invention, the association of disease-resistance or tolerance genes with molecular markers will render positional cloning possible (Kilian, A.; Chen, J.; Han, F.; Steffenson, B.; Kleinhofs, A. (1997). Towards map-based cloning of the barley stem rust resistance genes Rpg1 and Rpg4 using rice as an intergenomic cloning vehicle. Plant Molecular Biology, v. 35, p. 187-195; Yahiaoui, N.; Srichumpa, P.; Dudler, R.; Keller, B. (2004). Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat. Plant Journal, v. 37, p. 528-538), and with cloning, the structure, organization and operation of genes can be better understood (Yan, P.; Chen, X. M. (2006). Molecular mapping of a recessive gene for resistance to stripe rust in barley. Theoretic Applied Genetics, v. 113, p. 529-537). Furthermore, this association enables the molecular marker-assisted screening to indirectly identify individuals in segregating populations (Kelly, J. D.; Gepts, P.; Miklas, P. N.; Coyne, D. P. (2003). Tagging and mapping of genes and QTL and molecular marker-assisted selection for traits of economic importance in bean and cowpea. Field Crops Research, v. 82, p. 135-154) carrying the favorable allele under selection, resulting in time and resource savings.
The methods of the present invention are also extremely important in backcrossing, accelerating the recovery of recurrent genotypes. It also enables the understanding of genome evolution and the different relationships between the different genes.
Furthermore, with the data provided by the present invention the gene pyramiding process will be facilitated. Gene pyramiding is a work-intensive process due to the difficulty in identifying the presence of multiple genes, since selection is done phenotypically, via the analysis of symptoms. Using molecular markers linked to the genes to be pyramided, it will be possible to monitor the genes introduced during the process (Alzate-Marin, A. L.; Cervigni, G. D. L.; Moreira, M. A. (2005). Marker-assisted selection in the development of disease resistant plants, with emphasis on common bean and soybean. Fitopatologia Brasileira, v. 30, n. 4, p. 333-342).
Definitions
The definitions below are included in order to clarify certain terms used in the scope of the invention so as to facilitate understanding:
Alleles: One or more alternative forms of a gene.
Chromosome: a discrete unit of the genome carrying many genes. Each chromosome consists of a very long molecule of duplex DNA and an approximately equal mass of proteins. It is visible as a morphological entity only during cell division.
Artificial crossing: A crossing to introduce a new genetic material into a plant with a view to develop a new variety.
Test cross: involves crossing of an unknown genotype to a recessive homozygote so that the phenotype of the progeny corresponds directly to the chromosomes carried by the parents of unknown genotype.
Gene Map Distance: measured in cM (centimorgans)=percentage recombination (sometimes subject to adjustments).
Divergence: the percent difference in the nucleotide sequence between two related DNA sequences or in the amino acid sequence between two proteins.
Gene: (cistron) a segment of DNA that is involved in producing a RNA molecule that can be translated into a polypeptide chain; it also includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Genotype: the genetic constitution of a cell or organism.
F1 Generation: The first filial generation, produced by crossing two parental lines (homozygotes).
Linkage group: includes all gene loci that can be linked (directly or indirectly) by linkage relations; can be equivalent to a chromosome.
Heterozygote: an individual that has different alleles at a particular gene locus.
Homozygote: an individual that has the same alleles at a particular gene locus on homologous chromosomes.
Linkage: the tendency of some genes to be inherited together. It is the result of the location of genes on the same chromosome and is measured by the recombination among the loci.
Locus: the position on a chromosome that is occupied by a specific gene with a given trait; a locus can be also be occupied by any of the alleles of the gene.
Genetic map: map which shows the relative positions of gene loci forming the genome of an organism. The map is determined based on the joint inheritance of the loci. The distances among loci are calculated by their recombination frequency and are measured in cM.
Marker, Molecular Marker, Nucleic Acid Marker: relates to a nucleotide sequence used as a reference point that identifies a genetically linked locus. A marker can be derived from the genomic nucleotide sequence or from expressed nucleotide sequences (such as, for instance, from cDNA). In the context of this invention, this term can be associated with a specific marker or another gene locus (for instance, a locus related to disease resistance), wherein the marker pair or the marker and the second locus are genetically linked in the same linkage group; therefore, they are in linkage disequilibrium. In this respect, linkage disequilibrium is defined as any deviation from the frequencies expected independently, indicating the existence of an association among loci. Therefore, for the molecular markers to contribute to the inheritance and improvement studies, the population needs to be in linkage disequilibrium, otherwise the probability of a specific class of marker occurring would be independent of the segregation of the alleles of a given gene or QTL of interest, for instance.
Improvement: science directed to the genetic modification of living organisms.
Pathogen: organism that causes disease, infectious agent.
PI—Plant Introduction: plant genotype incorporated into any region different from its primary origin center.
Gene pyramiding: relates to the accumulation of two or more genes that yield the phenotype of interest in elite genotypes, either by classical improvement methods or by transformation.
Polymorphism: relates to the simultaneous occurrence in the population of genomes having allelic variations (such as alleles that yield different phenotypes or —for instance—the difference in the size of the sequences of certain microsatellites).
Tandem Repeats: multiple copies of the same sequence arranged in series.
Disease Resistance: genetic ability to prevent infection by a pathogen. Some forms of resistance operate by pathogen exclusion, some by preventing pathogen spread, and some by tolerating pathogen toxin; ability to resist to abiotic or biotic factors.
Backcrossing: a crossing of an individual with one of its parents. The descendants are called backcrossing generation or progeny.
Screening: describes the use of certain conditions to enable the survival of cells having a certain phenotype.
Susceptible: incapable of resisting to or tolerating damages caused by biotic or abiotic stress.
Tolerant: organism (plant) having the ability to live with a pathogen.